The objective of our research is to begin the development of a new treatment for deficiencies of the enteric nervous system (ENS) by transplanting ENS stem cells into the post-natal gut in such a way that they contribute to the ENS in vivo. Currently treatment generally involves resection of the affected bowel and morbidity remains high. The most common and best studied ENS deficiency is Hirschsprung disease, of which there are several animal models. We will use the rat model of Hirschsprung disease to establish this new treatment. [unreadable] [unreadable] Techniques now exist to prospectively identify and isolate stem cells of the ENS (neural crest stem cells, NCSCs) by flow cytometry and these cells can be isolated from the post-natal gut and from the proximal gut in animal models of Hirschsprung disease. We hypothesize that these cells can be used to build a distal ENS in individuals with Hirschsprung disease. To test this, we will inject genetically marked NCSC into the intestinal wall of young healthy or Hirschsprung rats. The animals will be allowed to recover. One week later, the intestine will be assessed for evidence of engraftment of the injected cells and their contribution to the neural and glial elements of the gut. Several important aspects of ENS development must be considered in this process. The developmental behavior of ENS progenitors is significantly influenced by molecules expressed by the gut mesenchyme which shows regional variation. Further, ENS progenitor cell-cell contact is required for migration. Therefore, our first specific aim is to determine the optimal cell density and location of NCSC injection for engraftment. [unreadable] [unreadable] The known gene defects causing Hirschsprung disease all involve signaling defects with the ENS precursor. Thus, the same gene defect that led to Hirschsprung disease may prevent successful NCSC autotransplantation to the aganglionic gut. Therefore, our second specific aim is to alter NCSC gene expression in such a way as to improve survival, proliferation, differentiation and migration of NCSC in isolated culture and in culture with intestinal segments. We will specifically examine how altered expression of genes implicated in Hirschsprung disease (specifically RET and EDNRB) affects NCSC behavior. We will manipulate gene expression in NCSC through infection with a retrovirus simultaneously producing green florescent protein and another gene of interest. This will alter gene expression not only in the NCSC but in all of its daughter cells. [unreadable] [unreadable] [unreadable]